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The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line.

Goshima G, Vale RD - J. Cell Biol. (2003)

Bottom Line: Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins.As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism.From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

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Roles of microtubule-based motors in the sequential steps of mitosis in S2 cells. Proper bipolar spindle assembly requires Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] (left pathway). Once a bipolar spindle is formed, chromosomes congress to the metaphase plate by the redundant actions of four chromosomal kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], Nod [Kid], and Klp67A [Kip3]. If a cell fails to assemble a bipolar spindle initially and a monopolar spindle is formed, it can be eventually converted to monastral bipolar spindle by chromatin-directed, acentrosomal pole-focusing mechanism that requires Klp61F [BimC/Eg5] and possibly Ncd [Kin C] functions (right pathway). After establishment of metaphase, Dhc64C [DHC] (cytoplasmic dynein) controls the timing of anaphase onset. Anaphase B spindle elongation is caused by sliding of antiparallel microtubules, which may require the functions of certain motor proteins that were not identified in our screening. Finally, central spindle formation and cytokinesis requires Pav [MKLP1].
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fig8: Roles of microtubule-based motors in the sequential steps of mitosis in S2 cells. Proper bipolar spindle assembly requires Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] (left pathway). Once a bipolar spindle is formed, chromosomes congress to the metaphase plate by the redundant actions of four chromosomal kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], Nod [Kid], and Klp67A [Kip3]. If a cell fails to assemble a bipolar spindle initially and a monopolar spindle is formed, it can be eventually converted to monastral bipolar spindle by chromatin-directed, acentrosomal pole-focusing mechanism that requires Klp61F [BimC/Eg5] and possibly Ncd [Kin C] functions (right pathway). After establishment of metaphase, Dhc64C [DHC] (cytoplasmic dynein) controls the timing of anaphase onset. Anaphase B spindle elongation is caused by sliding of antiparallel microtubules, which may require the functions of certain motor proteins that were not identified in our screening. Finally, central spindle formation and cytokinesis requires Pav [MKLP1].

Mentions: Our results provide several general insights into the roles of molecular motors in mitosis. First, cells appear to have redundant or alternate mechanisms for completing mitosis, despite the absence of a kinesin motor. Our experiments, for example, uncovered a novel rescue mechanism for converting monopolar to bipolar spindles by an acentrosomal pole-focusing mechanism (discussed below). By performing multiple RNAi, we also could show that several kinesin act in a partially redundant manner to ensure congression of chromosomes to the metaphase plate. Only three kinesins appear to be absolutely essential for completing mitosis; Klp61F [BimC/Eg5] and Klp67A [Kip3] RNAi cells could not proceed into anaphase, and Pav [MKLP1]–depleted cells could not execute cytokinesis. The phenotypes from our comprehensive RNAi analysis enable us to derive a model for the roles of microtubule-based motors in the sequential steps of mitosis in S2 cells (Fig. 8), the details of which will be described in the following paragraphs.


The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line.

Goshima G, Vale RD - J. Cell Biol. (2003)

Roles of microtubule-based motors in the sequential steps of mitosis in S2 cells. Proper bipolar spindle assembly requires Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] (left pathway). Once a bipolar spindle is formed, chromosomes congress to the metaphase plate by the redundant actions of four chromosomal kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], Nod [Kid], and Klp67A [Kip3]. If a cell fails to assemble a bipolar spindle initially and a monopolar spindle is formed, it can be eventually converted to monastral bipolar spindle by chromatin-directed, acentrosomal pole-focusing mechanism that requires Klp61F [BimC/Eg5] and possibly Ncd [Kin C] functions (right pathway). After establishment of metaphase, Dhc64C [DHC] (cytoplasmic dynein) controls the timing of anaphase onset. Anaphase B spindle elongation is caused by sliding of antiparallel microtubules, which may require the functions of certain motor proteins that were not identified in our screening. Finally, central spindle formation and cytokinesis requires Pav [MKLP1].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172859&req=5

fig8: Roles of microtubule-based motors in the sequential steps of mitosis in S2 cells. Proper bipolar spindle assembly requires Klp61F [BimC/Eg5], Ncd [Kin C], Klp10A [Kin I], and Klp67A [Kip3] (left pathway). Once a bipolar spindle is formed, chromosomes congress to the metaphase plate by the redundant actions of four chromosomal kinesins, CENP-meta [CENP-E], Klp3A [chromokinesin], Nod [Kid], and Klp67A [Kip3]. If a cell fails to assemble a bipolar spindle initially and a monopolar spindle is formed, it can be eventually converted to monastral bipolar spindle by chromatin-directed, acentrosomal pole-focusing mechanism that requires Klp61F [BimC/Eg5] and possibly Ncd [Kin C] functions (right pathway). After establishment of metaphase, Dhc64C [DHC] (cytoplasmic dynein) controls the timing of anaphase onset. Anaphase B spindle elongation is caused by sliding of antiparallel microtubules, which may require the functions of certain motor proteins that were not identified in our screening. Finally, central spindle formation and cytokinesis requires Pav [MKLP1].
Mentions: Our results provide several general insights into the roles of molecular motors in mitosis. First, cells appear to have redundant or alternate mechanisms for completing mitosis, despite the absence of a kinesin motor. Our experiments, for example, uncovered a novel rescue mechanism for converting monopolar to bipolar spindles by an acentrosomal pole-focusing mechanism (discussed below). By performing multiple RNAi, we also could show that several kinesin act in a partially redundant manner to ensure congression of chromosomes to the metaphase plate. Only three kinesins appear to be absolutely essential for completing mitosis; Klp61F [BimC/Eg5] and Klp67A [Kip3] RNAi cells could not proceed into anaphase, and Pav [MKLP1]–depleted cells could not execute cytokinesis. The phenotypes from our comprehensive RNAi analysis enable us to derive a model for the roles of microtubule-based motors in the sequential steps of mitosis in S2 cells (Fig. 8), the details of which will be described in the following paragraphs.

Bottom Line: Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins.As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism.From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.

Show MeSH